Assessment of Systemic Delivery of rAAVrh74.MHCK7.micro-dystrophin in Children With Duchenne Muscular Dystrophy: A Nonrandomized Controlled Trial

Abstract

Importance: Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7).

Objective: To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD.

Design, setting, and participants: This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (≥12 weeks).

Interventions: A single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion).

Main outcomes and measures: Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes.

Results: Four patients were included (mean [SD] age at enrollment, 4.8 [1.0] years). All adverse events (n = 53) were considered mild (33 [62%]) or moderate (20 [38%]), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events [50%]). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year.

Conclusions and relevance: This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care.

Trial registration: ClinicalTrials.gov Identifier: NCT03375164.

Conflict of interest statement

Conflict of Interest Disclosures: Dr Mendell reported receiving grants from Parent Project Muscular Dystrophy; receiving personal fees from Sarepta Therapeutics Inc., and Nationwide Children's Hospital outside the submitted work; and holding a pending patent to micro-dystrophin cassette for gene therapy and an issued patent to rAAV.SGCA delivery isolated limb infusion. Dr Sahenk reported receiving grants from Sarepta Therapeutics Inc during the conduct of the study. Ms Lehman reported receiving grants from Sarepta Therapeutics Inc. during the conduct of the study. Ms Nease reported receiving grants from Nationwide Children's Hospital during the conduct of the study. Ms Nicholl reported receiving grants from Nationwide Children's Hospital during the conduct of the study. Dr Al-Zaidy reported receiving consulting fees from AveXis Inc outside the submitted work. Dr Lewis reported being an employee of Sarepta Therapeutics Inc. during the conduct of the study. Dr Potter reported receiving other from Sarepta Therapeutics Inc. during the conduct of the . Ms Griffin reported receiving other from Sarepta Therapeutics Inc. during the conduct of the study. Dr Pozsgai reported receiving other from Sarepta Therapeutics Inc. during the conduct of the study and outside the submitted work. Dr Dugar reported being an employee of Sarepta Therapeutics Inc. Dr Rodino-Klapac reported receiving personal fees from Myonexus Therapeutics outside the submitted work, holding a patent (pending, licensed, and with royalties paid) to adeno-associated virus delivery of muscle-specific micro-dystrophin to treat patients with muscular dystrophy, and being an employee of Sarepta Therapeutics Inc. No other disclosures were reported.

Figures

Figure 1.. Study Flow Diagram

Figure 2.. Lack of Vector-Associated Toxic Effects by Histopathological Assessments in 4 Patients

A, Hematoxylin-eosin staining (original magnification ×20) shows unremarkable alterations in central nucleation (yellow arrowheads) of muscle fibers before and after treatment. Baseline (pretreatment) biopsy results show levels of necrosis (white arrowheads), stages of degeneration or regeneration, and increased connective tissue (black arrowheads). B, Picrosirius red staining shows a reduction in fibrosis after treatment. C, Fibrosis in tissue samples was measured by the percentage of collagen accumulation in tissue section. The error bars refer to the SD.

Figure 3.. Systemic Administration of rAAVrh74.MHCK7.micro-dystrophin

A, Frozen biopsied tissue sections of the gastrocnemius muscle were processed and stained with DYS3 antibody to detect micro-dystrophin. B, Micro-dystrophin compared with full-length protein was seen in 4 patients with Duchenne muscular dystrophy (DMD) before and after treatment (day 90) with rAAVrh74.MHCK7.micro-dystrophin. α-Actinin was used as a loading control, and sample from a patient was included as a negative control. NC indicates normal control. aPatient 4 had a positive immunoreactive band that was above the upper limit of quantification and was diluted 1 to 4 before loading in the representative gel.